Metagenomic Analysis of Fever, Thrombocytopenia and Leukopenia Syndrome (FTLS) in Henan Province, China: Discovery of a New Bunyavirus Bianli Xu1.*, Licheng Liu2., Xueyong Huang1., Hong Ma1, Yuan Zhang3, Yanhua Du1, Pengzhi Wang2, Xiaoyan Tang1, Haifeng Wang1, Kai Kang1, Shiqiang Zhang4, Guohua Zhao4, Weili Wu3, Yinhui Yang5, Haomin Chen1, Feng Mu2, Weijun Chen2,3,5* 1 Center for Disease Control and Prevention of Henan Province, Zhengzhou, China, 2 Beijing Genomics Institute in Wuhan, Wuhan, China, 3 Key Laboratory of Genome Sciences and Information, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China, 4 Center for Disease Control and Prevention of Xinyang City, Xinyang, China, 5 State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing, China Abstract Since 2007, many cases of fever, thrombocytopenia and leukopenia syndrome (FTLS) have emerged in Henan Province, China. Patient reports of tick bites suggested that infection could contribute to FTLS. Many tick-transmitted microbial pathogens were tested for by PCR/RT-PCR and/or indirect immunofluorescence assay (IFA). However, only 8% (24/285) of samples collected from 2007 to 2010 tested positive for human granulocytic anaplasmosis (HGA), suggesting that other pathogens could be involved. Here, we used an unbiased metagenomic approach to screen and survey for microbes possibly associated with FTLS. BLASTx analysis of deduced protein sequences revealed that a novel bunyavirus (36% identity to Tehran virus, accession: HQ412604) was present only in sera from FTLS patients. A phylogenetic analysis further showed that, although closely related to Uukuniemi virus of the Phlebovirus genus, this virus was distinct. The candidate virus was examined for association with FTLS among samples collected from Henan province during 2007–2010. RT-PCR, viral cultures, and a seroepidemiologic survey were undertaken. RT-PCR results showed that 223 of 285 (78.24%) acute-phase serum samples contained viral RNA. Of 95 patients for whom paired acute and convalescent sera were available, 73 had serologic evidence of infection, with 52 seroconversions and 21 exhibiting a 4-fold increase in antibody titer to the virus. The new virus was isolated from patient acute-phase serum samples and named Henan Fever Virus (HNF virus). Whole-genome sequencing confirmed that the virus was a novel bunyavirus with genetic similarity to known bunyaviruses, and was most closely related to the Uukuniemi virus (34%, 24%, and 29% of maximum identity, respectively, for segment L, M, S at maximum query coverage). After the release of the GenBank sequences of SFTSV, we found that they were nearly identical (.99% identity). These results show that the novel bunyavirus (HNF virus) is strongly correlated with FTLS. Citation: Xu B, Liu L, Huang X, Ma H, Zhang Y, et al. (2011) Metagenomic Analysis of Fever, Thrombocytopenia and Leukopenia Syndrome (FTLS) in Henan Province, China: Discovery of a New Bunyavirus. PLoS Pathog 7(11): e1002369. doi:10.1371/journal.ppat.1002369 Editor: Gustavo Palacios, George Mason University, United States of America Received March 14, 2011; Accepted September 28, 2011; Published November 17, 2011 Copyright: ß 2011 Xu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was sponsored by Infectious Diseases Special Project, Minister of Health of China (2008ZX10004–001-B, 2009ZX10004–103, 2009ZX10004– 109), Henan Medical Science Project (200702016) and China-Australia Health and HIV/AIDS Facility (EID35). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] (BX); [email protected] (WC) . These authors contributed equally to this work. Introduction clinical characteristics as the case definition to search for similar cases in local hospitals in this and neighboring counties, while In May 2007, a county hospital in Xinyang City, Henan establishing a disease surveillance system that required all medical Province treated three patients with fever, abdominal pain, institutions to report cases that met the above case definition. bloating, nausea, vomiting, gastrointestinal bleeding, and elevated Altogether, 79 cases were found in 2007 in Henan, with 10 aminotransferases. The local hospital diagnosed the disease as fatalities (case fatality rate, 12.7%). All patients were farmers and acute gastroenteritis. A family member of one patient reported the resided in mountainous or hilly villages, and many had reported disease to the Henan Center for Disease Control and Prevention tick bites 7–9 days before illness, further suggesting an infectious (CDC), which sent a team to investigate. The investigation etiology. In recent years, patients with similar clinical symptoms revealed that the disease had the following characteristics: (1) acute were reported with human granulocytic anaplasmosis (HGA; onset with fever; (2) low white blood cell and platelet counts; (3) Anaplasma phagocytophilum) in neighboring Anhui province [1]. In high levels of alanine and aspartate transaminases; (4) positive 2005, there was an epidemic of Tsutsugamushi (scrub typhus/ urine protein. On the basis of these features, the Henan CDC Orientia tsutsugamushi) in this area [2]. Clinical investigations, excluded the possibility of gastrointestinal disorders. In order to epidemiological analyses, and laboratory testing prompted con- identify the disease etiology, the Henan CDC team used the above sideration of rickettsial diseases as possible causes, including HGA, PLoS Pathogens | www.plospathogens.org 1 November 2011 | Volume 7 | Issue 11 | e1002369 Metagenomic Analysis: Discovery of a New Bunyavirus Author Summary tion methods, and 5) electron microscopic study of the morphology of the cultured virus. Initially in 2007, and again between 2008 and 2010, cases of Culture followed by serological and molecular tests is a standard a life-threatening disease with sudden fever, thrombocyto- approach for identifying an unknown virus. However, culture of penia, and leukopenia were reported in Henan Province, an unknown virus is time-consuming, even taking several years to China. Patient reports of tick bites suggested that this confirm a novel infection like HIV [17]. Otherwise, virus culture disease could be infectious or tick-transmitted. Many often fails because of the lack of cell lines capable of supporting patients were provisionally diagnosed with human granu- propagation of viruses (e.g., hepatitis B and C virus). Methods for locytic anaplasmosis (HGA). However, only 24 of 285 (8%) cloning nucleic acids of microbial pathogens directly from clinical had objective evidence of HGA, suggesting that other samples offer opportunities for pathogen discovery, thereby laying pathogens likely contributed to fever, thrombocytopenia and leukopenia syndrome (FTLS). Illumina sequencing was the foundation for future studies aimed at assessing whether novel used for direct detection in clinical samples of pathogens or unexpected viruses play a role in disease etiology. Random possibly associated with FTLS. A novel bunyavirus was PCR and subtractive cloning sequencing have identified previ- found only in samples from FTLS patients. Further ously unknown pathogens as etiological agents of several acute and epidemiologic and laboratory investigation confirmed that chronic infectious diseases [18,19]. Recently, high-throughput the novel bunyavirus was associated with FTLS. The results sequencing approaches have been used for pathogen detection and illustrate that metagenomic analysis is a powerful method discovery in clinical samples [20–22]. We also developed a method for the discovery of novel pathogenic agents. Combined for exploring viruses, both known and novel, using high- with epidemiologic investigation, it could assist in rapid throughput Illumina sequencing. In this study, high-throughput diagnosis of unknown diseases and distinguish them from Illumina sequencing was applied to specifically explore the viral other diseases with similar symptoms caused by known communities in patients with FTLS, using healthy subjects as pathogens. controls. Here, we provide evidence for the discovery of a novel bunyavirus associated with FTLS through high-throughput sequencing. Subsequent culture of the virus and PCR detection human monocytic ehrlichiosis (HME; Ehrlichia chaffeensis), and of the specific virus in patient specimens confirmed these findings. Tsutsugamushi disease. Specific methods such as polymerase chain reaction (PCR) and immunofluorescence assays (IFAs) for these Materials and Methods pathogens were then used to determine if these cases were attributable to HGA or HME [3,4]. However, only 18 of 79 Ethics statement (22.7%) patients were positive for A. phagocytophilum based on This research was approved by the Review Board of the Center serology and DNA testing. Thus, the disease was initially for Disease Control and Prevention of Henan Province, the considered at least partly caused by A. phagocytophilum, and cases Review Board of the Center for Disease Control and Prevention of were provisionally diagnosed as suspected HGA based on clinical Xinyang city,